4-octyl itaconate alleviates cisplatin-induced ferroptosis possibly via activating the NRF2/HO-1 signalling pathway

Abstract

Ferroptosis, characterized by iron-dependent lipid reactive oxygen species (ROS) accumulation, plays a pivotal role in cisplatin-induced ototoxicity. Existing research has suggested that in cisplatin-mediated damage to auditory cells and hearing loss, ferroptosis is partially implicated. 4-Octyl itaconate (4-OI), derived from itaconic acid, effectively permeates cell membranes, showcasing potent anti-inflammatory as well as antioxidant effects in several disease models. Our study aimed to investigate the effect of 4-OI on cisplatin-induced ferroptosis and the underlying molecular mechanisms. The survival rates of HEI-OC1 cells and mice cochlea hair cells were measured by CCK8 and immunofluorescence, respectively. The auditory brainstem response (ABR) audiometry was used to detect changes in hearing thresholds in mice before and after treatment. Levels of ROS were evaluated by DCFH-DA. Real-time PCR quantified inflammatory cytokines TNF-α, IL-6 and IL-1β. Network Pharmacology and RNA sequencing (RNA-seq) analysis of the potential mechanism of 4-OI resistance to cisplatin-induced ferroptosis. The expressions of ferroptosis-related factors (GPX4, SLC7A11 and PTGS2) and important antioxidant factors (NRF2, HO-1, GCLC and NQO1) were tested by real-time PCR, Western blot and immunofluorescence. Results demonstrated cisplatin-induced significant ROS and inflammatory factor release, reduced NRF2 expression, hindered nuclear translocation and activated ferroptosis. Pretreatment with 4-OI exhibited anti-inflammatory and antioxidant effects, along with resistance to ferroptosis, ultimately mitigating cisplatin-induced cell loss. In the present study, we show that 4-OI inhibits cisplatin-induced ferroptosis possibly through activation of the NRF2/HO-1 signalling pathway, thereby exerting a protective effect against cisplatin-induced damage to auditory cells, and providing a new therapeutic strategy for cisplatin-induced hearing loss.

Keywords: 4-octyl itaconate; NRF2; cisplatin; ferroptosis; ototoxicity.

FIGURE 1

4‐OI alleviates the cisplatin‐induced decrease in HEI‐OC1 cell viability. (A) Cell survival rate by CCK8 assay after 24 h treatment of different concentrations of cisplatin (0, 10, 20, 30, 60 and 80 μM) on HEI‐OC1 cells. (B) CCK8 assay of cell survival after treatment of 30 μM cisplatin applied to HEI‐OC1 cells for different treatment times (0, 3, 6, 12, 24 and 48 h). (C) Cell survival rate detected by CCK8 assay after 24 h of 4‐OI treatment at different concentrations on HEI‐OC1. (D) Cell survival rate based on CCK8 of HEI‐OC1 cells first treated with different concentrations of 4‐OI for 3 h and then by 30 μM cisplatin for 24 h. The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05, **p < 0.01 and ****p < 0.0001 versus control group, respectively, and by ^#### p < 0.0001 versus cisplatin group. ‘ns’ indicates no significance; ctl, control; Cis, cisplatin.

FIGURE 2

4‐OI mitigates cisplatin‐induced reduction in cochlear hair cell survival. (A, B) Representative images of Myosin 7a (green) and DAPI (blue) in the apical, middle and basal turns of the cochlear basement membrane with immunofluorescence staining in five groups of samples. (C–E) Number of Myosin 7a positive hair cells per 100 μm at the apical, middle and basal turns of the cochlear basement membrane in the five groups of samples shown in (A). (F–H) Number of Myosin 7a positive hair cells per 100 μm at the apical, middle and basal turns of the cochlear basement membrane in the five groups of samples shown in (B). The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus control group, respectively, and by ^## p < 0.01 and ^### p < 0.001 versus cisplatin group, respectively. ‘ns’ marks no significance. Scale bar = 40 μm. Cis, cisplatin.

FIGURE 3

4‐OI reduces cisplatin‐induced ROS and inflammatory factors production and accumulation. (A) Four groups of samples stained for DCFH‐DA in live cells with DCFH‐DA used as a measure of intracellular ROS. (B) Quantitative analysis of DCFH‐DA immunofluorescence intensity of HEI‐OC1 living cells shown in (A). (C) Representative images of four groups of samples of the cochlear basement membrane transferred to DCFH‐DA (green) and Hoechst (blue) staining. (D) Quantitative analysis of DCFH‐DA immunofluorescence intensity in cochlear basement membrane shown in (C). (E–G) Relative mRNA levels of TNF‐α, IL‐6 and IL‐1β in four groups of HEI‐OC1 cells. The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, respectively. Scale bar = 40 μm. Cis, cisplatin.

FIGURE 4

Cisplatin inhibits the NRF2 signal pathway and induces ferroptosis in vitro. (A–D) Relative mRNA levels of NRF2, SLC7A11, GPX4 and PTGS2 in five groups of HEI‐OC1 cells treated with cisplatin for 0, 3, 6, 12 and 24 h, respectively. (E) Protein expressions of NRF2, SLC7A11 and GPX4 in five groups of HEI‐OC1 cells. (F–H) Quantitative statistics of NRF2, SLC7A11 and GPX4 proteins shown in (E). The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 versus control group, respectively. ‘ns’ indicates no significance. Cis, cisplatin.

FIGURE 5

Network pharmacological analysis of the association of 4‐OI with ferroptosis. (A) Venn diagram of 4‐OI targets predicted with ferroptosis driver genes and ferroptosis suppressor genes. FRGs, ferroptosis related genes. (B) Protein–protein interaction (PPI) network of intersecting targets ranked according to degrees. (C) Two subnetworks obtained by the MCODE plugin in Cytoscape. (D) GO terms of biological process based on GO enrichment analysis of the intersecting genes. (E) KEGG enrichment analysis of the intersecting genes. (F) Molecular docking of 4‐OI with BRD2. (G) Molecular docking of 4‐OI with KEAP1.

FIGURE 6

Differentially expressed genes (DEGs) and their KEGG and GO enrichment analyses. (A) Volcano plot of all DEGs presented in blue dots and red dots for down‐ and up‐regulated genes, respectively. (B) Heatmap of clustering of ferroptosis‐related genes presented in red for relatively high expression and blue for relatively low expression. (C–E) GO enrichment analysis based on DEGs with genes annotated in GO terms of (C) biological process (BP), (D) cellular component (CC) and (E) molecular function (MF), respectively. (F) KEGG enrichment analysis based on DEGs.

FIGURE 7

4‐OI inhibits cisplatin‐induced ferroptosis. (A–C) Relative mRNA levels of SLC7A11, GPX4 and PTGS2 in four groups of HEI‐OC1 cells. (D) Western blot analysis of SLC7A11 and GPX4 in four groups of HEI‐OC1 cells, with cisplatin used for modelling. (E, F) Quantitative statistics of SLC7A11 and GPX4 proteins shown in (D). (G) Western blot analysis of SLC7A11 and GPX4 in four groups of HEI‐OC1 cells, with RSL3 used for modelling. (H, I) Quantitative statistics of proteins SLC7A11 and GPX4 shown in (G). (J) GPX4 immunofluorescence images of four groups of HEI‐OC1 cells (GPX4 and DAPI double staining). (K) Representative images of the cochlear basement membranes of four groups of samples transferred to Myosin 7a (green), GPX4 (red) and DAPI (blue) immunofluorescence staining. The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, respectively. Scale bar = 40 μm. Cis, cisplatin.

FIGURE 8

4‐OI alleviates cisplatin‐induced damage to HEI‐OC1 cells and cochlear hair cells by activating NRF2/HO‐1 signalling pathway. (A–D) Relative mRNA levels of NRF2, HO‐1, NQO1 and GCLC in four groups of HEI‐OC1 cells. (E) Western blot analysis of NRF2, HO‐1 and NQO1 in four groups of HEI‐OC1 cells. (F–H) Quantitative statistics of NRF2, HO‐1 and NQO1 proteins shown in (E). (I, J) Immunofluorescence images of NRF2 and HO‐1 in four groups of HEI‐OC1 cells (double staining of NRF2, HO‐1 and DAPI). (K) Western blot analysis of NRF2 of cytosolic and nuclear origins in four groups of HEI‐OC1 cells. (L) Representative images of immunofluorescence staining of Myosin 7a (green), NRF2 (red) and DAPI (blue) in the middle turn of the cochlear basement membrane in four groups of samples. The values are expressed as mean ± SEM (n = 3). The significant differences are determined by *p < 0.05 and **p < 0.01, respectively. ‘ns’ indicates no significance. Scale bar = 40 μm. Cis, cisplatin.

FIGURE 9

Protective effect of 4‐OI against cisplatin‐induced hearing loss in adult C57BL/6J mice. (A) The specific dosing regimen of 4‐OI and cisplatin was administered via intraperitoneal injection in 8‐week‐old adult male mice. (B) The auditory brainstem response (ABR) thresholds in mice from four experimental groups following pure tone stimulation. (C) Representative images of Myosin 7a (green) in the apical, middle and basal turns of the cochlear basement membrane based on immunofluorescence staining in four groups of samples. (D–F) Number of Myosin 7a positive hair cells per 100 μm at the apical, middle and basal turns of the mice cochlear basement membrane in the four groups of samples shown in (C). The values are expressed as mean ± SEM (n = 6). The significant differences are determined by *p < 0.05, ***p < 0.001 and ****p < 0.0001, respectively. ‘ns’ indicates no significance. Scale bar = 40 μm. Cis, cisplatin.